Effects of Sirolimus treatment on patients with β‐Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4 + and CD8 + T cells

Abstract Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β‐Thalassemia). The objective of the present study was to analyse the activity of CD4+ and CD8+ T cells in β‐Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN‐γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4+ and CD8+ T cells, respectively. The main results of the present study are that treatment of β‐Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4+ and CD8+ T cells releasing IFN‐γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β‐Thalassemia patients are considered prone to immune deficiency.


| INTRODUC TI ON
Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed by many studies to improve vaccine responses, especially in elderly. This was for instance reported to impact on the Flu vaccine efficacy. 1,2 Accordingly, testing mTOR inhibitors and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. [3][4][5] Among the effects of mTOR inhibitors responsible for improving the vaccine activity, enhanced CD8 + effector memory T-cell function has been proposed. 6,7 Collectively, there is agreement on the fact that mTOR inhibitors might play an important role in boosting vaccination of elderly and fragile people as well as memory T-cell function in general. [3][4][5][8][9][10] In this respect, Sirolimus (also known as rapamycin, a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus) 11,12 is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases, such as renal, cardiac and liver transplantation, [13][14][15][16] systemic lupus erythematosus, 17 lymphangioleiomyomatosis, 18 tuberous sclerosis complex, 19 recurrent meningioma, 20 pancreatic neuroendocrine tumours, 21 advanced differentiated thyroid cancers, 22 advanced breast cancer, 23 diffuse large B-cell lymphomas 24 and metastatic renal cell carcinoma. 25 Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia and sickle-cell disease). [26][27][28][29][30][31] The β-Thalassemias are due to more that 300 hereditary mutations of the β-globin gene, inducing absence or low-level synthesis of β-globin in erythroid cells. [32][33][34] It is widely accepted that high production of foetal haemoglobin (HbF) is beneficial for β-Thalassemia patients. [35][36][37][38] In a recent study, we have reported biochemical, molecular and clinical results of the Sirolimus-based NCT03877809 clinical trial Sirthalaclin). 39,40 The rationale of this trial was that Sirolimus is of interest in β-Thalassemia, since it induces the expression of foetal haemoglobin (and this might contribute to ameliorate the clinical parameters of these patients), induces autophagy (thereby reducing the excess of free α-globin) and, finally, might contribute to mobilization of erythroid cell from the bone marrow (thereby reducing anaemia).
The results of the trial were obtained in 8 patients with β + /β + and β + /β 0 genotypes, treated with a starting dosage of 1 mg/day Sirolimus for 24-48 weeks. 40 The first finding of the study was that expression of γ-globin mRNA was increased in blood and erythroid precursor cells isolated from β-Thalassemia patients treated with low-dose Sirolimus. A second important conclusion was that Sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess of free α-globin chains, bilirubin, soluble transferrin receptor and ferritin). In most of the patients, a decrease of the transfusion demand index was observed.
Altogether, the data obtained suggested that Sirolimus given at low doses modifies haematopoiesis and induces increased expression of γ-globin genes in a subset of β-Thalassemia patients.
The major end points of the NCT03877809 clinical trial were not aimed to explore changes in the functionality of memory T cells of Sirolimus-treated patients. 39 However, the possible effect of Sirolimus in β-Thalassemia is intriguing, as recent reports have highlighted a high mortality rate in β-Thalassemia patients affected by infectious bacterial and viral diseases, [41][42][43][44][45][46] probably due to co-existing immune deficiencies. [47][48][49] Immune dysfunctions characterizing thalassemia patients include changes in lymphocyte subsets, such as the accumulation of suppressor T cells and the reduced proliferative capacity and numbers of T helper cells, as well as the defective activity of natural killer (NK) cells. Similarly, an altered humoral immunity has been shown in patients with β-Thalassemia. 48 Therefore, efficient vaccination is of primary importance and molecules exhibiting boosting effects on vaccines should be considered of great relevance.
The objective of the present study was to analyse the activity of CD4 + and CD8 + T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples from the NCT03877809 clinical trial. The approach was to verify IFNγ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to CEF and CEFT peptide pools, that activate CD8 + and CD4 + T cells, respectively.

| Patient recruitment and treatment with Sirolimus
Recruitment of the Sirthalaclin pilot clinical trial and data collection (EudraCT n° 2018-001942-33, NCT 03877809) was at the Thalassemia Centre of Azienda Ospedaliera-Universitaria S.Anna, as extensively reported in Zuccato et al. 40 All the patients were transfusion-dependent (TDT, Transfusion Dependent Thalassemia).

| Autophagy detection in memory T cells subpopulations
PBMCs were treated as above mentioned in the analysis of the immunophenotype section and stained with an autophagosome tracker (Cyto-ID) and a different antibody mix in order to detect autophagy levels in different memory T cells subpopulations inside CD4 + and CD8 + lymphocytes (see Table 3). Cells were stained with Cyto-ID autophagy detection kit 2.0 (Enzo LifeSciences) following manufacturer protocol and then washed in PBS and stained with LIVE/Dead™ Fixable Aqua-Dead Cell Stain Kit and incubated 10′ in the dark. After an additional wash step, cells were stained with the antibody mix showed in Table 3 and incubated 15′ in the dark.

| Analysis of cytokines, chemokines and growth factors
Plasma samples were isolated from β-Thalassemia patients by direct centrifugation of the whole blood, as described in Zuccato et al. 40 Proteins were measured using Bio-Plex Human Cytokine 27-plex Assay (Bio-Rad) as suggested by the manufacturer and described in Gasparello et al. 53 and in the Supplementary Materials (SM1).
The assay allows the multiplexed quantitative measurement of 27

| Statistical analysis
Unless otherwise stated, all the data were normally distributed and presented as mean ± S.D. Statistical differences between groups were compared using Wilcoxon test or one-way anova (analyses of variance between groups) for repeated measures followed by Dunnet post hoc tests. Statistical differences were considered significant when p < .05 (*), highly significant when p < .01 (**).

Sirolimus-treated β -Thalassemia patients
Immunophenotyping of PBMCs was performed by flow cytometry analysing the following markers associated with the various lym-

| Effects of Sirolimus on naive and memory CD4+ and CD8+ T cells subpopulations
As depicted in the representative experiment shown in Figure 2, the analyses performed have been implemented for obtaining F I G U R E 1 Summary representing the % of regulatory T lymphocytes (Treg) in respect to the total number of CD4 + T lymphocytes (A). (B-C) Summary representing the % of CD4 + and CD8 + T lymphocytes, respectively, on live PBMCs. D. Ratios of CD4 + /CD8 + T cells. A complete summary of the immunophenotype is reported in Figure S2

TA B L E 3
List of antibodies employed for T memory Lymphocyte subset typing information on the following CD4 + ( Figure 3A) and CD8 + ( Figure 3B Figure 3B).
Sirolimus, through mTOR inhibition, could increase autophagy levels, 54,55 and studies on humans and mice suggest that higher rates of autophagy sustain the long-term survival of memory T cells. 10,56 For instance, experimental mice lacking the autophagy gene Atg7 in T cells failed to establish memory CD8 + T cells specific for influenza and MCMV infection. 56 To investigate whether Sirolimus treatment affected autophagy levels in different T-cell subsets, PBMCs isolated at Day 0 or after at least 90 days after treatment with Sirolimus were analysed for the contents of autophagic vesicles by flow cytometry.
As shown in Figure 4, we observed a slight, although not nonsignificant, trend towards higher autophagy levels in both central memory CD4 + ( Figure 4A) and CD8 + ( Figure 4B) T cells, while autophagy levels of the other T-cell subsets were unchanged.

| Effects of Sirolimus treatment on activity of memory CD4+ T cells
The functionality of CD4 + T cells was analysed by the ELISpot tech-

| Effects of Sirolimus on activity of memory CD8+ T cells of β -Thalassemia patients
The activity of memory C8 + T cells was analysed following stimulation of the same isolated PBMCs employed for CD4 + memory T-cell activity but stimulating them with the CEF peptide pool instead. The CEF peptide pool contains 32 MHC class I-restricted viral peptides from human CMV, EBV and Influenza virus and can selectively stimulate epitope-specific human CD8 + T cells to produce IFNγ.
The data obtained are presented and summarized in Figure 6. The data obtained indicate that after three or more months of Sirolimus treatment, each patient showed a marked increase in the number of epitope-specific memory CD8 + T cells releasing IFNγ following stimulation with CEF peptide pool (p = .031). Importantly, IL-1β was the only pro-inflammatory cytokine whose levels were augmented by Sirolimus (see for instance the unaltered levels of MIP-1α or TNFα, reported in Figure S3), suggesting a reversion of immunosuppression rather than the induction of a lowgrade inflammatory state.
Together, these data suggest that Sirolimus could systemically restore the cytokine milieu of β-Thalassemia patients dampening Th2 responses and immune suppression environments without provoking excessive inflammation levels.

| DISCUSS ION
The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity of memory CD4 + and CD8 + T cells. This conclusion is outlined by the data depicted in Figures 5 and 6, that indicate  Figure S3, respectively). *p < .05, **p < .01 suppressor T cells and the reduced proliferative capacity and numbers of T helper cells, as well as the defective activity of natural killer (NK) cells. Similarly, an altered humoral immunity has been shown in patients with β-Thalassemia. 48 Accordingly, β-Thalassemiaassociated immunosuppression should be actively targeted to protect these patients.
In Of interest is also the decrease of IL-13 ( Figure 7B, p = .043) and IP-10 ( Figure 7D, p = .008) in Sirolimus-treated patients. Concerning IL-13, it has been reported that blocking IL-13 enhances the therapeutic effect of antigen-specific immunotherapy by enforcing the activity of CD41 T cells. 69 Concerning IP-10, it is established that high levels of this protein decrease T-cell function. 70 The inhibitory effects of Sirolimus on IL13 and IP-10 sustain the concept that this mTOR inhibitor might divert helper cells towards Th1, rather than Th2, polarization and enhance T-cell function, as indicated by the experiments reported in Figures 5 and 6.
Finally, a remarkable decrease of G-CSF was observed ( Figure 7C, p = .008). Interestingly, G-CSF has been demonstrated to be a strong immune regulator of T cells, being C-CSF treatment able to potently reduce reactivity of T cells and imbalance the Th1/Th2 ratio. 58 This exploratory experimental plan should stimulate further studies on the effects of Sirolimus on immunomodulation, especially considering the pro-inflammatory status of β-Thalassemia patients. 74 Our data do not clarify whether the effect of Sirolimus was mediated directly on memory T cells or indirectly on APC. This cannot be in theory excluded, as it is known that autophagy processes regulate peptide presentation in professional antigen-presenting cells, which mediate thymocyte selection. 67 Nonetheless, our secretome data tend to suggest that Sirolimus can impact on the cytokine milieu, and this translates in increased levels of EM CD4 + T cells and of Th1 CD4 and CD8 recall responses.
In any case, and whatever being the mechanism of action, the beneficial effect of Sirolimus treatment on COVID-19 vaccination might be of great interest for managing β-Thalassemia, as an increased mortality risk from COVID-19 was observed in Italian patients affected by hemoglobinopathies, including β-Thalassemia. 75 A further issue in β-Thalassemia is premature ageing of the immune system, that has been suggested to affect the response to SARS-CoV-2 mRNA vaccine. 76 Considering that autophagy and mitophagy in T cells decrease with age, 10 the induction/potentiation of these biological processes in elderly should be considered of great importance.

ACK N O WLE D G E M ENTS
The authors would like to thank Dr. Maria Rita Gamberini (Day Hospital Thalassemia, Arcispedale S. Anna, Ferrara) for support and helpful discussion.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

PATI E NT CO N S E NT FO R PU B LI C ATI O N
All patients signed an informed consent, that includes their consent to publication.